The proposed experiments are part of a long-term plan to investigate the genetic control of nervous system remodeling during development. Because of the combined power of molecular and classical genetic approaches to developmental problems, Drosophila CNS metamorphosis has been chosen as an experimental model system. Steroid hormones influence many aspects of CNS development in vertebrates and invertebrates. Determining the mechanisms mediating hormonal effects on CNS morphogenesis, as well as on neuronal birth, differentiation, and death, are critical to understanding abnormalities of human nervous system development. In keeping with the known action of steroid hormones on gene expression, mutants of a gene regulated by the steroid molting hormone 20- hydroxyecdysone (20HE) exhibit specific defects in regional CNS morphogenesis and neural organization. this gene, the Broad Complex (BR- C), is expressed in many tissues, including the CNS, and regulates the activities of many other loci. The proposed experiments are based on the hypothesis that CNS defects in the mutants result from the misregulation of BR-C target genes in the CNS. Thus, the major goal of this project is the isolation and characterization of 20HE-inducible genes expressed in the CNS, with particular emphasis on those regulated by the BR-C. Initially, BR-C transcription in the CNS will be studied by in situ hybridization and confocal microscopy. The structure and hormone- inducibility of these transcripts will be analyzed by standard RNA blotting and mapping methods. The scheme for isolating hormone-inducible genes from CNS RNA combines the time-honored approach of differential hybridization with newer cDNA cloning vectors which allow in vitro synthesis of cRNA. This amplified cRNA provides high-sensitivity reagents for library construction and analysis. A subtracted cDNA library, enriched for sequences preferentially expressed in hormone-exposed CNS tissue, will be constructed and screened. A variety of criteria, including misregulation in BR-C mutants, will be used to determine which 20HE-inducible clones represent genes with probable important roles during CNS metamorphosis. The expression patterns and DNA sequence of these genes will be determined and their function assessed by the generation and analysis of mutants.